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Bump-and-hole engineering of human polypeptide N-acetylgalactosamine transferases to dissect their protein substrates and glycosylation sites in cells.

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posted on 2023-01-16, 14:31 authored by Beatriz Calle, Edgar Gonzalez-Rodriguez, Keira E Mahoney, Anna Cioce, Ganka Bineva-Todd, Omur Y Tastan, Chloe Roustan, Helen Flynn, Stacy A Malaker, Benjamin Schumann
Despite the known disease relevance of glycans, the biological function and substrate specificities of individual glycosyltransferases are often ill-defined. Here, we describe a protocol to develop chemical, bioorthogonal reporters for the activity of the GalNAc-T family of glycosyltransferases using a tactic termed bump-and-hole engineering. This allows identification of the protein substrates and glycosylation sites of single GalNAc-Ts. Despite requiring transfection of cells with the engineered transferases and enzymes for biosynthesis of bioorthogonal substrates, the tactic complements methods in molecular biology. For complete details on the use and execution of this protocol, please refer to Schumann et al. (2020)1, Cioce et al. (2021)2, and Cioce et al. (2022)3.

Funding

Crick (Grant ID: CC1068, Grant title: STP Structural Biology) Crick (Grant ID: CC1063, Grant title: STP Proteomics) Crick (Grant ID: CC2127, Grant title: Schumann CC2127)

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